InhibitionofhepatitisBvirus(HBV)byLNA-mediatednuclearinterferencewithHBVDNAtranscription.doc

精品论文inhibition of hepatitis b virus (hbv) by lna-mediated nuclear interference with hbv dna transcription5sun zhen, chen hongyan, lu daru(school of life science,fudan university)abstract: silencing target genes with small regulatory rnas is widely used to investigate gene function and therapeutic drug development. recently,triplex-based approaches have provided anotherattractive means to achieve targeted gene regulation and gene manipulation at the molecular and10cellular levels. nuclear entry of oligonucleotides and enhancement of their afnity to the dna targetsare key points of such approaches. in this study, we developed lipid-based transport of alocked-nucleic-acid (lna)-modied oligonucleotide for hepatitis b virus (hbv) dna interference in human hepatocytes expressing hbv genomic dna. in these cells, the lna-modied oligonucleotides passed efciently across the cell membrane, and lipid-coating facilitated translocation from the15cytoplasm to the nucleus. the oligonucleotide specifically targeting hbv dna clearly interfered withhbv dna transcription as shown by a block in pregenomic rna (pgrna) production. the hbv dna-targeted oligonucleotide suppressed hbv dna replication and hbv protein production moreefciently than small interfering rnas directed to the pgrna. these results demonstrate that fusionwith lipid can carry lna-modied oligonucleotides to the nucleus where they regulate gene expression.20interfering with hbv dna transcription by lna-mod-ied oligonucleotides has strong potential as anew strategy for hbv inhibition.key words: rna interference; hepatocyte; oligonucleotide; lipofectamine; triple-helix0introduction25rna-guided gene silencing uses double-stranded rna (dsrna) to inhibit gene expression in a sequence-specic manner. in rna interference (rnai), dsrna is processed into small interfering rnas (sirnas), which guide the cleavage of complementary rna molecules by forming the rna-induced silencing complex 1,2. although rnai predominantly takes place in the cytoplasm, rnai in the nucleus has long been predicted and sirna was recently shown to be transported into the nucleus by specic proteins 3. in addition to30the investigation of gene function, rnai has also been exploited for wider applications such as treatment of viral diseases 4,5.through binding in the major groove of double-stranded dna at oligopyrimidineoligopurine sequences, triple-helix-forming oligonucleotides (tfos) interfere with gene transcription, replication and recombination 6,7.the use of these oligonucleotides is becoming an attractive means to achieve targeted gene regulation and gene35manipulation both in vitro and in vivo 8,9. in addition, modi cation of tfos with locked nucleic acid (lna)nucleotides, analogs of dna nucleotides that contain a methylene linkage between the 20 oxygen and the 40 carbon of the ribose ring, enhance triplex stability and improve the intracellular ef ciency of tfos 1012. lna-containing oligonucleotides have been used for antisense gene inhibition 13,14.hepatitis b virus (hbv) is a 3.2 kb dna virus, which uncoats its nucleocapsid in the cytoplasm of human40hepatocytes and delivers its genome into the cell nucleus where the hbv dna is converted into a covalently closed circular dna (cccdna) 15,16. the cccdna is the template for all the viral mrna, including the pre-genomic rna (pgrna) which serves as the template for core protein and polymerase synthesis. it has recently been shown that expression of short hairpin rna (shrna) homologous to hbv mrna inhibits hbv replication effectively in cultured cells and mammalian liver 17,18. nevertheless, the current use of rnai mainly impedes45the translation of hbv mrna in the cytoplasm rather than antagonizing the viral dna in the nucleus. although suppression of the polymerase and core protein synthesis as well. as disturbance in pgrna packaging can prevent virus replication, it is reasonable to assume that a block in upstream hbv dna transcription would be moreeffective. therefore, we set out to design an lna-modi ed hbv dna-targeted oligonucleotide as a potential new approach for hbv suppression.foundations: this study was supported by grants from the state s&t pro-jects of china (2008zx10002-007) to zhi chen and by the na-tional natural science foundation of china (30771090) to wei liu. the project also sponsored by the scientic research foundation for the returned overseas chinese scholars, state educationministry, supported by doctoral fund of ministry of education of china（20090071120）.brief author introduction:sun zhen(1982),male,geneticscorrespondance author: lu daru(1965),male,professor,genetics. e-mail: - 9 -501experimental procedures1.1 oligonucleotides, cell culture, and cell transfectionhea lna-oligonucleotides probed with alex546 at the 50-terminal were synthesized by iba-naps (iba). hepg2.2.15 cells were used in all experiments. they were maintained in dmem supplemented with 10% fbs and 400 lg/ml g418, at 37 oc in a 5% co2 incubator.55transient transfection of probed lna-oligonucleotides was conducted with or withoutlipofectamine 2000 reagent (invitrogen) according to the manufacturers instructions.1.2 fluorescence microscopycell imaging was performed in the multitracking mode on a laser scanning confocal microscope (lsm510, carl zeiss) with a 63x plan apochromat 1.4 na objective. at 4 h after60transfection, the cells in labtek chambers (nalge nunc international) were stained with dapi (1 lg/ml) for 5 min, then the culture medium was replaced by dmem and the cells were imaged using a sequential scan setting with excitation at 405 and 453 nm. emission was collected at420480 nm (dapi) and 560615 nm (alexa546).1.3 quantitative analysis of supernatant viral proteins65hbsag and hbeag protein levels in the culture medium were analyzed using a time-resolveduoro-immuno assay (peirce) according to the manufacturers protocol.1.4 western blottingcells were washed twice with ice-cold pbs and lysed in lysis buffer (50 mm hepes, ph 7.5,0.5% nonidet p-40, 150 mm nacl, 1 mm edta, 1 mm egta, 1 mm sodium orthovanadate, 170mm dithiothreitol, 1 mm naf, 2 mm phenylmethylsulfonyl uoride and 10 mg/ml each ofpepstatin, leupeptin, and aprotinin). soluble protein (15 mg) was separated by sdspage and transferred to nitrocellulose membrane (whatman). primary antibodies against hbcag (diluted1:2000, santa cruz) and gapdh (diluted 1:5000, santa cruz) were used. the complexes were treated with enhanced chemiluminol reagent (santa cruz) and visualized on x-ray lm (kodak).751.5 qrt-pcranalysis of hbv replication intermediates was performed using quantitative real-time pcr. briey, the viral dna in the culture medium was extracted with a minibest viral dna extraction kit (takara). the cellular rnas underwent cytoplasmic extraction with trizol (invitrogen). then the lysates were treated with deoxyribonuclease i (invitrogen) to remove the80dna. after phenol/chloroform purication, the encapsulated viral rna was precipitated in ethanol. qrt-pcr was performed using sybr* premix ex taq reagents and a one step sybr* primescript rt-pcr kit (takara). primers used for hbv dna and rnaquantication were: nc_03977: 1823-1846 nt: ttt ccc ctc tgc cta atc atc tca; 22932316 nt: ata ccc gca ttt ggt ggt ctg taa. primers used for mrna nor-malization were, gapdh, bc083511:85395-416 nt: gga gcc aaa agg gtc atc atc t; 606-626 nt: agg ggc cat cca cag tct tct.1.6 northern blottingnorthern blot analysis was carried out using the agarose-form-aldehyde method. about 15 lg of rna per sample was separated on 1.5% agarose-formaldehyde gel and then upward capillary transferred to a biodyne b nylon membrane (pierce). hbv tran-scripts and endogenous control90gaphd were detected using chemiluminescent probes. all the probes were labeled with a biotin random prime labeling kit (pierce). the primers used to generate pgrna and gapdh hybrid probes were: nc_003977: 1996-2016 nt: cac cgc ctc tgc tct gta tcg; 2292-2315 nt: tag ggg cat ttg gtg gtc tgt aag; bc083511: 395-416 nt: gga gcc aaa agg gtc atc atc t; 606-626 nt: agg ggc cat cca cag tct tct.952results1001051101151201251301352.1 lipofectamine facilitates the nuclear entry of lna-oligonucleotidessearching for a method to suppress nuclear transcription of hbv, we designed threelna-modied oligonucleotides probed with alex546 at the 50-terminal. oligonucleotides p1 and p2 were antisense and targeted two different areas of the pgrna, and p3 was a sense sequence of pgrna targeting the same region of hbv dna in the nucleus (fig. 1a and b). we added the lna-modied oligonucleotides to hepg2.2.15 cells, a human hepatoma cell line transfected with cloned hbv dna and expressing all the hbv genomic genes 19. then we observed byconfocal microscopy that in a few hours, the lna-oligonucleotides appeared in the cells and were distributed predominantly in the cytoplasm (fig. 1c). surprisingly, when they were pre-cultured with lipofectamine and then added to the cells, oligonucleotides accumulated dramatically in the nuclei, as demonstrated by a perfect colocalization with the nuclear marker dapi (fig. 1d). these results therefore show that these lna-oligonucleotides are efciently taken up by cells and lipofectamine markedly enhances their translocation from the cytoplasm to the nucleus.2.2 nuclear dna-targeted lna-oligonucleotide inhibits hbv protein synthesisgiven that lipofectamine facilitated the nuclear translocation of lna-oligonucleotides, we then investigated their effects on hbv gene expression. first, we measured the hbeag and hbsag proteins secreted into the culture medium. at 24 and 48 h after transfection, p2 showed very little effect on protein expression. p1 moderately inhibited the production of hbeag with or without lipofectamine (fig. 2b and d), indicating a major antagonism to cytoplasmic pgrna. p3 lowered both the hbsag and hbeag protein levels only in the presence of lipofectamine andhad much higher activity than p1 (fig. 2a and b), suggesting a nuclear inhibition of hbv dna. in the presence of lipofectamine, hbsag in the medium was moderately reduced not only by p3 but also by p1 (fig. 2a and c), even though the oligonucleotides were not designed to target the hbsag coding region. a possible explanation for this is that the p3 covered the region of the hbsag transcription factor binding site and p1 displayed a partial inhibition of nuclear hbv dna.to examine the expression level of nucleocapsid hbcag protein, which is required for hbv viral replication, total cell lysates were immunoblotted after culturing the cells with the oligonucleotides for 24 h. as a result, with or without lipofectamine, p1 and p2 signicantly decreased the intracellular hbcag level (fig. 2e). at the same time, p3 had a strong inhibitory effect on hbcag expression in lipofectamine-based culture, but only weak suppression in the absence of the lipid (fig. 2e), suggesting a major nuclear effect of p3 on hbv dna transcription.2.3 nuclear dna-targeted lna-oligonucleotide inhibits hbv gene transcription to conrm the inhibitory effect of the lna-modied oligonucleotides in the nucleus, we quantied the cellular hbv pgrna and hbv genomic dna in the cell culture medium usingreal-time pcr. we found a slight decline in hbv dna level brought about by p1 inlipofectamine-free culture (fig. 3a), while both p1 and p3 dramatically reduced the dna level in the presence of lipofectamine (fig. 3b), clearly demonstrating a nuclear effect. further, p1 treatment partially reduced the cellular hbv pgrna and this reduction was enhanced by lipofectamine (fig. 3c and d). in p3-treated cells, hbv pgrna decreased to a great degree only in the presence of lipofectamine (fig. 3c and d). northern hybridization showed that p3 induceda140145fig.1. design and intracellular localization of lna-oligonucleotides. (a) schematic representation of hbv transcripts. the hbv open reading frames are shown aligned with the 3.5 kb pgrna/core mrna. the targeting sites of designed antisense oligonucleotides p1 and p2, and the sense oligonucleotide p3 are indicated. (b) sequences of the oligonucleotides. antisense oligonucleotides p1 and p2 were designed to complement two separate sequences of hbv pgrna. the lnas are show in red while the dnas are shown in blue, which are also the cleavage sites for rnase h. sense oligonucleotide p3 was designed as the sense sequence of the sa me region of p1 and p2 in pgrna. the lnas (red) were mixed with the dnas (blue) at specic sites to avoid the formation of hairpins or dimers. (c and d) intracellular distribution of the oligonucleotides. hepg2.2.15 cells were transfected with 5 nm alexa-543-labeled lna-oligonucleotides in the absence (c) or presence (d) of lipofectamine, and the cells were imaged by confocal microscopy 4 h after transfection. blue, nucleus stained with dapi; red, alexa-543-labeled lna-oligonucleotide. all oligonucleotides showed the same type of localization, so only the results of p3 are represented. (for interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)150155fig. 2. sense lna-oligonucleotide inhibits hbv protein expression. (ad) hbsag (a and c) and hbeag (b and d) protein levels in the culture medium were measured by time-resolved uoro-immunoassay at 24 h (a and b) or 48 h (c and d) after transfection with 5 nm lna-oligonucleotides in the presence (black symbols) or absence (white symbols) of lipofectamine. (e) cellular hbcag protein levels were determined by western blot at 24 h after transfection, where the intracellular gapdh was used as a loadingcontrola clear depletion of mature pgrna in the presence of lipofectamine (fig. 3e). these results suggest specic antagonistic effects of lna-modied oligonucleotides on nu clear hbv dna transcription. p3 prevented pgrna production by targeting hbv dna in the nucleus while p1 mainly caused pgrna degradation.160165170175fig. 3. sense lna-oligonucleotide silences hbv genes. (ad) hepg2.2.15 cells were transfected with 5 nm anti-sense p1 or sense p3lna-oligonucleotide in the absence (a and c) or presence (b and d) of lipofectamine. hbv dna in the cell culture medium (a and b) and hbv pgrna in the cell lysates (c and d) were quantied by real-time pcr at indicated times after transfection. white symbols: blank control; lled symbols: p1; black symbols: p3. (e) quantication of pgrna by northern blot. the gure represents a reproducible result of three independent experiments in hepg2.2.15 cells in which the cells were transfected with p3 with or without lipofectamine and were tested 24 h after transfection. endogenous gapdh was used as control for loading integrity of the total rna.3discussionin this study, we have shown that an lna-oligonucleotide designed to target nuclear hbv dna efciently suppresses hbv replication in cultured hepatic cells.so far, dna triple-helix-based approaches to control and modulate gene functions at the level of genomic dna have suffered from the lack of afnity of triplex-forming oligonucleotides for their dna targets. oligonucleotides based on the lna sugar unit can greatly enhance triplex stability, which partly alleviates the sequence restriction constraints 10,12,20,21. our data indicate that, once in the nucleus, a mixed lna and dna oligonucleotide can effectively target an integrated hbv dna sequence, thereby inhibiting hbv replication at the transcription level. although the sugar unit of lna could change the distribution of molecular electro-negativity, making entry to the nucleus easier, our results demonstrated that coating with lipid dramatically enhanced the entry of180185190195lna-oligonucleotides to the nucleus, challenging the traditional view of nuclear delivery of nucleotides. it has long been thought that cell uptake is a relatively inefcient process and once inside the cell, the nucleotide-lipid complex has no inherent mechanism to deliver the nucleotides to the nucleus. prior to entering the nucleus, oligonucleotides would be released from the plasmidlipid complex, and freely diffuse toward the nuclear pores, the aqueous channels of the nucleus 20. however, a recent study has shown in caenorhabditis elegans, a specic argonaute protein nrde-3 is required to transport specic classes of small regulatory rnas into the nucleus for gene expression regulation 3. therefore, it will be interesting to clarify whether a similar transport system occurs in other species, and investigate the interactions between nrde-3 and nucleotides in the presence or absence of lipofectamine.together with a previous report in animals 21, our results suggest that lna-modied oligonucleotides can pass through the plasma membrane of cultured cells even without using transfection machinery. considering that hepg2.2.15 is a strain that is very difcult for gene transfection, modication of an oligonucleotide with lna woul